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Side-chain oxidized cholesterol derivatives, like 25-hydroxycholesterol (25-OH-Chol) are important regulators of cellular cholesterol homeostasis. How transport of oxysterols through the endo-lysosomal pathway contributes to their biological function is not clear. The Niemann-Pick C2 protein (NPC2) is a small lysosomal sterol transfer protein required for export of cholesterol from late endosomes and lysosomes (LE/LYSs). Here, we show that 25-hydroxy-cholestatrienol, (25-OH-CTL), an intrinsically fluorescent analogue of 25-OH-Chol, becomes trapped in LE/LYSs of NPC2-deficient fibroblasts, but can efflux from the cells even in the absence of NPC2 upon removal of the sterol source. Fluorescence recovery after photobleaching (FRAP) of 25-OH-CTL in endo-lysosomes was rapid and extensive and only partially dependent on NPC2 function. Using quenching of NPC2's intrinsic fluorescence, we show that 25-OH-Chol and 25-OH-CTL can bind to NPC2 though with lower affinity compared to cholesterol and its fluorescent analogues, cholestatrienol (CTL) and dehydroergosterol (DHE). This is confirmed by calculations of binding energies which additionally show that 25-OH-CTL can bind in two orientations to NPC2, in stark contrast to cholesterol and its analogues. We conclude that NPC2's affinity for all sterols is energetically favored over their self-aggregation in the lysosomal lumen. Lysosomal export of 25-OH-Chol is not strictly dependent on the NPC2 protein.

Summary: The efforts addressed in this thesis refer to applying nonlinear risk predictive techniques based on logistic regression to medical diagnostic test data. This study is motivated and pursued to address the following: 1. To extend logistic regression model of biostatistics to medical informatics 2. Computational preemptive and predictive testing to determine the probability of occurrence (p) of an event by fitting a data set to a (logit function) logistic curve: Finding upper and lower bounds on p based on stochastical considerations 3. Using the model developed on available (clinical) data to illustrate the bounds-limited performance of the prediction. Relevant analytical methods, computational efforts and simulated results are presented. Using the results compiled, the risk evaluation in medical diagnostics is discussed with real-world examples. Conclusions are enumerated and inferences are made with directions for future studies., by Alice Horn Dupont., Thesis (M.S.C.S.)--Florida Atlantic University, 2011., Includes bibliography., Electronic reproduction. Boca Raton, Fla., 2011. Mode of access: World Wide Web.